This research revolves around the development of laser scanning microscopy as a quantitative tool for cell biology and biophysics. To that end, the first three year grant was devoted to design and construction of a flexible instrument which would serve researchers as well as be a proving ground for new developments in scanned imaging. The goals of this phase of the project have nearly been achieved, and this competing continuation proposal addresses further scientific use of the instrument and several advanced technological issues related to the integration of optical design and digital postprocessing in the microscope system. The specific aims of this continuation proposal are (1) To refine the optical and electronic design of the scanner and detector systems, in order to maximize signal-to-noise ratio with minimum optical dose to the specimen. (2) To use the laser scanning microscope in scientific investigations (neutrophil activation responses and cognate activation of B lymphocytes), to develop practical quantitative methods and test the instrument's performance; (3) To continue development of digital image processing methods for tomographic reconstruction from noisy (photon-limited) confocal image sets; (4) To investigate the fundamental limits and practical realization of so-called superresolving pupils for enhancing lateral or longitudinal resolution in the confocal microscope; and (5) To develop an integrated model for the microscope system which elucidates the roles played by optics and digital postprocessing, and which enables an optimum system to be designed. The results of this work will be applicable to other laser scanning microscopes, as well, and hence will further the general state of the art in scanning microscopy.